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Animal Studies Safeguard Food
Supplies—andHuman Health

A cow naturally infected with M. avium subspecies
paratuberculosis that is in the late stages of disease and has typical
clinical signs such as weight loss, watery diarrhea, and general poor health.
This cow is part of a study herd used in research on Johne’s
disease at the National Animal Disease Center, Ames, Iowa.
(D1441-1)

Hundreds of different species of Mycobacterium are
known to exist, and many of them have been infecting animals and
humans—sometimes with deadly results—for thousands of years.
Scientists at the Agricultural
Research Service’s National Animal Disease Center (NADC) in Ames,
Iowa, are fighting back against two of its most damaging
variants—Johne’s disease and bovine tuberculosis (bovine TB).

M. avium subspecies paratuberculosis (MAP) is
responsible for the onset of Johne’s disease, which results in losses
exceeding $200 million every year to the U.S. cattle industry. Experts believe
that almost 70 percent of U.S. cattle herds are infected with MAP, and an
animal can spread the disease soon after it is infected. But noticeable
symptoms—including severe weight loss and diarrhea—can take up to 5
years to develop.

“MAP is like a stealth organism,” says NADC
microbiologist Judy Stabel. “It shelters in the host’s white blood
cells and stays at low levels until stress makes the disease apparent. And
it’s one of the hardest organisms to work with in the field.”

“We need a better way to detect infected animals early
on,” adds NADC microbiologist John Bannantine.

Microbiologist Judy Stabel bottle feeds young calves involved
in a project to evaluate immune responses to a vaccine that protects against
Johne’s disease.
(D1442-1)

Protein Prospecting

The MAP genome—which contains all its genes—has
been sequenced, so researchers now have information about the different
proteins that are made from those genes. Bannantine and colleagues took some of
this genetic data and assembled an array of 96 proteins. They wanted to
identify proteins that might be useful in confirming a diagnosis of
Johne’s disease or that might be targeted for therapeutic intervention.

The researchers used the 96 proteins in the array to identify
which ones prompted the most robust immune response from antibodies in the
serum of infected cattle. They found three proteins that consistently drew the
strongest attacks from serum antibodies—a level of immune response that
clearly linked the three proteins with the onset of the disease. Bannantine
believes that with additional work, these segments might provide crucial
building blocks for development of a diagnostic tool for Johne’s disease.

“This protein array is the only one like it in the
world,” Bannantine says. “Because we’ve been so careful in
selecting the MAP proteins for our array, we’re confident that the
antibodies are responding to MAP proteins—not similar proteins produced
by other Mycobacterium species.”

Bannantine is also very pleased that their studies have cleared
up another aspect of MAP infection.

“When an animal is first infected, there is a
cell-mediated response to the bacterium,” he says. “We thought that
another type of immune response—the one that produces
antibodies—developed much later. But in experimentally infected animals,
we can use this array to detect exposure to MAP as early as 70 days after the
animal is infected, much earlier than previously reported in field
studies.”

The next step is to determine whether these early-detected
antigens are recognized by infected cattle in real-world dairy herds. “We
also need to determine the extent of cross-reactivity these proteins have with
other environmental mycobacteria, because one problem with current
Johne’s disease tests is the lack of specificity,” Bannantine says.

Stabel has been studying more about the early stages of the
cell-mediated response to MAP and finding ways to diagnose the disease in young
animals. “We’ve found a way to use information about the
cell-mediated response to detect MAP in naturally infected calves that are only
6 months old,” she says. “When animals this young are diagnosed,
then the producer can decide how best to respond—either by removing the
animal from the herd or looking at other options.”

Stabel has also helped evaluate animal models for MAP research
and has concluded that a smaller ruminant model—like goats or
sheep—shows promise. “These animals are slightly quicker to reach a
clinical disease state,” she says.

Two of the deer maintained at the Agricultural Research
Service's National Animal Disease Center in Ames, Iowa. The unique research
herd is used to study vaccines for prevention of tuberculosis in white-tailed
deer.(D1445-1)

New Approaches to an Ancient Disease:
Tuberculosis

The USDA Animal and Plant Health Inspection Service (APHIS)
tests more than 1 million animals every year for bovine TB. A lot can ride on
the outcome. For instance, from May 2002 through June 2004, 875,616 cows from
687 California herds were tested for bovine TB. As a result, 13,000 cows were
culled.

During that time, dairy herd quarantines were enforced to
ensure that contaminated milk from infected cows did not enter the human food
supply and transmit the disease to people. This additional enforcement cost
individual producers as much as $70,000 per month in lost income.

Finding effective diagnostics and vaccines for bovine TB is
crucial for livestock and human health and producer profits. At NADC, around 80
white-tailed deer—some so tame that they try to steal food from the
pockets of their caretakers—are key to this effort.

“White-tailed deer are a significant reservoir of bovine
TB,” says NADC veterinary medical officer Mitch Palmer. “When the
deer jump fences and share livestock feed, they can contaminate the feed with
TB bacteria. That’s one way cattle herds become exposed to the disease
and become infected.”

Because of the threat wild deer pose to domestic cattle, Palmer
and his colleagues have been vaccinating white-tailed deer with the human TB
vaccine M. bovis BCG. They want to see whether vaccination could be
part of a successful effort to control the disease in wildlife.

“What we’ve found is that the vaccine is effective
in decreasing the severity of the disease and that oral vaccination appears to
be as effective as subcutaneous vaccination,” Palmer says. “But we
have some safety concerns. Because it’s a live vaccine that stays in body
tissues for up to 250 days, bacteria in the vaccine that prompt the protective
immune response might also infect humans who consume venison from vaccinated
deer. And vaccinated deer can shed the vaccine and expose other animals to the
disease.”

The vaccine research has yielded other significant findings as
well—including results that could affect human health.

NADC molecular biologist Tyler Thacker led studies indicating
that in white-tailed deer, levels of interferon gamma (IFN-gamma)—a
protein that is critical in the immune response to bovine TB
infection—increase as the severity of the infection increases. This
finding countered previous studies suggesting that levels of IFN-gamma rose
when the immune system of a vaccinated animal was successfully fighting the
pathogen.

Veterinary medical officers Ray Waters (left) and Mitch Palmer
(right) prepare to collect blood to be used in developing improved tests for
tuberculosis in cattle.(D1446-1)

Other results in the study suggested that elevated levels of
IL-4, another immune protein, could be found in animals with lower infection
levels. This also contradicted research with other animal species suggesting
that IL-4 levels increase with disease severity.

“These results give us more accurate information about
whether a trial vaccine is effective,” Thacker says. “Vaccine
testing is expensive, so it’s helpful to have screening tools for
interpreting results. Now that we know IFN-gamma is not a good predictor of
vaccine efficacy, we can start looking for other indicators.”

The NADC scientists have also been using neonatal calves to
test human TB vaccines. This approach is cheaper and safer to use than testing
in nonhuman primates.

“We’ve devised a very effective method for
inoculating neonatal calves with TB by using an aerosol challenge,” says
NADC veterinary medical officer Ray Waters. “This gives us new options
for testing human TB vaccines on animals with immune responses that closely
resemble the human immune response.”

In addition, the most common time for administering the vaccine
to humans is soon after birth—timing that is mimicked when neonatal
calves are used for tests. The neonatal calf tests are currently the most
effective bridge between testing in mice and nonhuman primates. Results have so
far been very similar to the findings in nonhuman primates.

Vaccines or Diagnostics—Which Comes
First?

“Vaccine development and diagnostic development are
intertwined,” Thacker observes. “If we find indicators that predict
immunity, then maybe we can refine those indicators and use them for diagnostic
testing as well.”

Thacker, Palmer, and Waters are working on improving bovine TB
tests for cattle. “We have serum-based tests that are not approved for
use in the TB eradication program yet,” Palmer says. “But they are
showing some potential for testing samples in the field, where time or
temperature could affect the stability of the blood samples.”

Meanwhile, producers and APHIS staff often use a commercial
blood-based test called “Bovigam” to confirm a preliminary positive
skin test diagnosis of bovine TB.

“Standardizing the protocol for using Bovigam is still a
work in progress,” says Waters. “Labs have been running different
test protocols, so we’re working on establishing standard measures for
processing blood samples. For instance, we’ve found that using vessel
plates to test samples is just as effective as using tubes, and with vessel
plates we don’t need to use as much of the reagent to obtain accurate
results. We also need to determine optimal schedules for collecting and
processing samples—and we need to determine a safe temperature range for
maintaining those samples before they reach the lab.”

Into the Wild

Animal health depends as much on meticulous fieldwork as it
does on painstaking lab studies. So Waters, Palmer, and Thacker were part of a
team that obtained blood samples from 760 wild deer and surveyed them for
bovine TB, the largest such sampling to date. The team found that the blood
tests accurately identified an infected animal about 70 percent of the time. On
the other hand, the tests correctly identified a noninfected animal about 90
percent of the time.

Palmer explains, “With most tests of this type,
there’s typically a tradeoff between the accuracy of positive and
negative results. We’re continuing to work on this because we’d
like to get both numbers in the high 80s or low 90s.

“We’re approaching bovine TB research from a range
of perspectives, but all of it helps us understand the immune response,”
Palmer continues. “When an animal is infected with TB, tissue destruction
is caused by the host reacting to the TB pathogen. We want to understand what
is causing the tissue destruction, which will help us find ways to treat the
disease and intervene in the process of tissue destruction.”—By
Ann
Perry, Agricultural Research Service Information Staff.

This research is part of Animal Health, an ARS national
program (#103) described on the World Wide Web at
www.nps.ars.usda.gov.

Rift Valley fever (RVF) was once confined to Africa—until
cases were confirmed in Saudi Arabia and Yemen in 2000. These cases sparked
concerns about the disease’s ability to spread further. RVF has never
reached the United States, but if it were to do so, researchers at the
Arthropod-Borne Animal Diseases Research Laboratory (ABADRL) in Laramie,
Wyoming, would be ready.

RVF is a potentially devastating zoonotic disease. Fortunately,
ABADRL scientists are collaborating with colleagues in the United States and
abroad to ensure that the nation will be prepared in the improbable event of a
U.S. outbreak.

Under the leadership of microbiologist William Wilson, ABADRL
scientists are pursuing RVF research with three main objectives: to determine
which North American mosquito species are competent to transmit the RVF virus;
to develop sensitive and specific diagnostic tools; and to develop and evaluate
vaccines.

To meet the first objective, ABADRL scientists are
investigating whether there is a vector genetic component to RVF transmission.
Preliminary studies have examined which mosquito and midge species are more
likely to act as virus vectors. The results suggest regional differences in the
ability of mosquito populations to become infected and transmit the virus. To
expand research in this area, ABADRL is increasing the size and scope of its
existing mosquito colonies to include additional species.

The ABADRL scientists are working with the Canadian Food
Inspection Agency and USDA’s Animal and Plant Health Inspection Service
to develop operator-safe, sensitive diagnostic tests for early detection. They
are also cooperating with the U.S. Department of Homeland Security to evaluate
the safety of potential commercial vaccines. ABADRL is collaborating with the
Canadian agency to develop livestock challenge models to evaluate the efficacy
of new vaccines as they are developed.

Such research ensures that the United States won’t be
caught off guard should an outbreak of RVF occur. The work also has immediate
practical applications in countries currently affected by the disease.By Laura McGinnis,
Agricultural Research Service Information Staff.

"Animal Studies Safeguard Food Supplies—andHuman Health" was published in the
May/June 2009
issue of Agricultural Research magazine.